Min-Jun Wang

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell‐derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell‐derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell‐derived DE cells, which were defined as Cxcr4+c‐Kit+, Cxcr4+E‐cadherin+ cells or Cxcr4+PDGFRa− cells, could be induced in the serum‐free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A‐mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell‐derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell‐derived hepatic cells and ES cell‐derived pancreatic cells for future regenerative medicine. J. Cell. Biochem. 112: 1022–1034, 2011.

Reversal of hepatocyte senescence after continuous in vivo cell proliferation

A better understanding of hepatocyte senescence could be used to treat age‐dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53‐p21 and p16ink4a‐pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase‐deficient (Fah−/−) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. Conclusion: These findings suggest that the hepatocyte “ploidy conveyer” is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy. (Hepatology 2014;60:349–361)

Hepatocyte polyploidization and its association with pathophysiological processes

A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

Murine embryonic stem cell-derived hepatocytes correct metabolic liver disease after serial liver repopulation

Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah(-/-)) mice. Albumin expressing (Alb(+)) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah(-/-) mice recipients. FAH expressing (FAH(+)) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH(+) hepatocytes increased significantly. ES cell-derived FAH(+) hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah(-/-) recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion, ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases.

Xeno-repopulation of Fah−/−Nod/Scid mice livers by human hepatocytes

Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics, infection of hepatitis viruses, and the efficacy of hepatitis vaccines. Significant levels of liver xeno-repopulation have been reported in Fah−/−Rag2−/−Il2rg−/− mice. However, the high mortality and low breeding rate of this model may hinder its application. A new model, termed Fah−/−Nod/Scid mice, which combines the advantages of liver repopulation in Fah−/− mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding. Fah−/−Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities. FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah−/−Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes; the proportion of engrafted human hepatocytes reached 33.6%. The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis, the expression of characteristic proteins, and the biochemical analysis of liver function. These results show that Fah−/−Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.

Altered Hepa1-6 cells by dimethyl sulfoxide (DMSO)-treatment induce anti-tumor immunity in vivo

Cancer immunotherapy is the use of the immune system to treat cancer. Our current research proposed an optional strategy of activating immune system involving in cancer immunotherapy. When being treated with 2% DMSO in culture medium, Hepa1-6 cells showed depressed proliferation with no significant apoptosis or decreased viability. D-hep cells, Hepa1-6 cells treated with DMSO for 7 days, could restore to the higher proliferation rate in DMSO-free medium, but alteration of gene expression profile was irreversible. Interestingly, tumors from D-hep cells, not Hepa1-6 cells, regressed in wild-type C57BL/6 mice whereas D-hep cells exhibited similar tumorigenesis as Hep1–6 cells in immunodeficient mice. As expected, additional Hepa1-6 cells failed to form tumors in the D-hep-C57 mice in which D-hep cells were eliminated. Further research confirmed that D-hep-C57 mice established anti-tumor immunity against Hepa1-6 cells. Our research proposed viable tumor cells with altered biological features by DMSO-treatment could induce anti-tumor immunity in vivo.

Insulin-like growth factor 2 is a key mitogen driving liver repopulation in mice

Hepatocyte transplantation holds great promise as an alternative to orthotopic organ transplantation in the treatment of liver diseases. However, obtaining clinically meaningful levels of liver repopulation has not been achieved because the mechanisms regulating hepatocyte proliferation in recipient livers have not yet been well characterized. In the mouse model of Hereditary Tyrosinemia Type I, the fumarylacetoacetate hydrolase-deficient (Fah−/−) mouse, we found gradually increasing expression level of insulin-like growth factor 2 (IGF2) in the hepatocytes of host livers. Similarly, high levels of IGF2 were found in the livers of patients with deficient FAH activity. Recombinant IGF2 directly promotes proliferation of primary hepatocytes in vitro. Inhibition on IGF2 expression through the interruption of PI3K/Akt and MAPK pathways significantly reduced the level of liver repopulation in Fah−/− mice. Interestingly, treatment with IGF2 before hepatocyte transplantation generally improved the amount of liver repopulation seen in various mice models of liver injury. Altogether, these findings underscore the underlying mechanisms of therapeutic liver repopulation in Fah−/− mice, and indicate that IGF2 is a potential hepatocyte mitogen for liver cell transplantation therapies.

Liver-enriched activator protein 1 as an isoform of CCAAT/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma

Purpose Liver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs. Materials and methods Differences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence. Results LAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell–associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo. Conclusion LAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

The extent of liver injury determines hepatocyte fate toward senescence or cancer

It is well known that induction of hepatocyte senescence could inhibit the development of hepatocellular carcinoma (HCC). Until now, it is still unclear how the degree of liver injury dictates hepatocyte senescence and carcinogenesis. In this study, we investigated whether the severity of injury determines cell fate decisions between hepatocyte senescence and carcinogenesis. After testing of different degrees of liver injury, we found that hepatocyte senescence is strongly induced in the setting of severe acute liver injury. Longer-term, moderate liver injury, on the contrary did not result into hepatocyte senescence, but led to a significant incidence of HCC instead. In addition, carcinogenesis was significantly reduced by the induction of severe acute injury after chronic moderate liver injury. Meanwhile, immune surveillance, especially the activations of macrophages, was activated after re-induction of senescence by severe acute liver injury. We conclude that severe acute liver injury leads to hepatocyte senescence along with activating immune surveillance and a low incidence of HCC, whereas chronic moderate injury allows hepatocytes to proliferate rather than to enter into senescence, and correlates with a high incidence of HCC. This study improves our understanding in hepatocyte cell fate decisions and suggests a potential clinical strategy to induce senescence to treat HCC.